YL0712-3, a Novel ATR/ATM Inhibitor, Demonstrates Anti-Tumor Activity in <i>TP53</i> Mutant DLBCL through Synthetic Lethality and Degradation of Mutant p53

Background: Patients with diffuse large B-cell lymphoma (DLBCL) harboring TP53 mutations (TP53mut DLBCL) face a dismal prognosis and currently lack a standardized treatment regimen. Both ATR (Ataxia Telangiectasia and Rad3-related protein) and ATM (Ataxia Telangiectasia Mutated) are reported to have synthetic lethal interactions with TP53, suggesting that inhibition of ATR/ATM could induce synthetic lethality in TP53 mutant tumor cells. Furthermore, mutant p53 proteins (mutp53) not only lose the tumor suppressive functions of wild-type p53 but also acquire gain-of-function (GOF) properties, making the degradation of mutp53 a promising therapeutic approach. Objective: This study aims to identify and evaluate the therapeutic efficacy and mechanisms of a novel ATM/ATR inhibitor, YL0712-3, targeting TP53 mutant DLBCL. Methods: A small molecule library was constructed based on proprietary core structure, from which we screened and further optimized a small molecule designated as YL0712-3. The kinase profile of YL0712-3 was screened and its downstream pathways (ATM/Chk2 and ATR/Chk1) were validated via Western Blot. The in vitro activity of YL0712-3 was assessed across various TP53 mutant and wild-type DLBCL cell lines, with ATR inhibitor Elimusertib and ATM inhibitor AZD1390 as positive controls. The in vivo efficacy of YL0712-3 was evaluated using cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. Additionally, YL0712-3 + R-CHOP regimen was evaluated in PDX mouse models. Mechanistic insights were investigated using co-immunoprecipitation (co-IP), Mass Spectrometry, and RNA sequencing. Results: YL0712-3 exhibited cytotoxic effects on both TP53 wild-type and mutant lymphoma cells, with significantly greater anti-tumor activity in TP53 mutant cells (IC50: 46.8nM vs. 299.2nM, P=0.0082). Kinase profiling indicated that YL0712-3 predominantly inhibits kinases in the PI3K and PIKKs families. Western blot analysis indicated that YL0712-3 predominantly inhibits the ATR/Chk1 pathway more strongly than the ATM/Chk2 pathway, with no significant inhibition of the PI3K/Akt pathway. In vitro, YL0712-3 demonstrated superior efficacy compared to AZD1390 and Elimusertib. The efficacy was also observed in PDX models. The combination of YL0712-3 with the R-CHOP regimen showed significant anti-tumor activity. Mechanistic studies revealed that YL0712-3 induces G1 phase arrest and apoptosis in SU-DHL-4 (TP53mut R273C, IC50=6.3nM) cells, and promotes mutant p53 protein degradation in a time- and dose-dependent manner. Co-IP, mass spectrometry, and RNA sequencing results suggest that this degradation involves the proteasome pathway. Conclusion: The ATR/ATM inhibitor YL0712-3 effectively targets TP53 mutant DLBCL cells by inhibiting the ATM/Chk2 and ATR/Chk1 pathways and inducing proteasome-mediated degradation of mutant p53 protein. YL0712-3 exhibits promising anti-tumor activity in both in vitro and in vivo models, especially when combined with R-CHOP, presenting a potential therapeutic strategy for TP53 mutant DLBCL.